The proposed research is concerned with five basic goals: a) to evaluate the role of the deoxynucleoside triphosphates in regulating DNA synthesis; b) to gain an understanding of how the network for supplying the deoxynucleoside triphosphate precursors of DNA functions in the intact cell; c) to evaluate the role of induced perturbations in deoxyribonucleoside pools in determining the mechanism of action and interaction of antimetabolites; d) to evaluate the relationship between imbalances in the deoxynucleoside triphosphate pools and the induction of cellular cytotoxicity (thymine-less death) and e) to evaluate the contributions of salvage pathways in vivo in altering the effectiveness and selectivity of antimetabolites. The first goal will be approached by continuing the kinetic analysis of mammalian DNA polymerases purified from calf thymus and human tumor cells and a determination of the regulatory properties of these enzymes with respect to the deoxynucleoside triphosphates. Further evaluation of the perturbations induced in both the ribonucleotide and the deoxyribonucleoside triphosphate pools by antimetabolites under conditions of variable steady-state growth and the evaluation of these results with a mathematical model of DNA synthesis should lead to further insight into the mechanism of action and interaction of these agents. The fourth goal will be approaches by determining the relationship between deoxynucleoside triphosphate pools, the appearance of single-strand breaks in DNA and the rate of cellular cytotoxicity as measured by cloning mamallian cells in the presence of different concentrations of drugs. Finally, attempts will be made in vivo to alter alter drug cytotoxicity and antitumor selectivity by supplying combinations of various drugs and salvage metabolites by continuous infusion in mice. Integration of the information obtained with these basic approaches should lead to a better understanding of the regulation of DNA synthesis and a more judicious use of antimetabolites both alone and in combination for clinical therapy.